Oxygenation of steroids in the 11beta position by spondylocladium



United States Patent OXYGENATION OF STEROIDS IN 11p POSITION BYSPONDYLOCLADIUM Joseph L. Sardinas, Brooklyn, and Gilbert M. Shull,Huntington Station, N. Y., assignors to Chas. Pfizer & Co., Inc.,Brooklyn, N. Y., a corporation of Delaware Application January 23, 1957Serial No. 635,600

7 Claims. (Cl. 195-51) No Drawing.

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and allopregnanes, G-dehydroprogesterone, esters of hydroxyl groups ofthese, etc.

The Spondylocladium genus belongs to the order Moniliales of the classFungi Imperfecti. Of particular value are strains of the species S.australe and S. xylogenum. Other microorganisms from the genus may beselected for conducting the process of this invention by simple testswhich will be described in more detail below. Many of these organismsare available in public culture collections and others may be isolatedfrom natural materials, such as soil, by standard procedures well knownto mycologists.

As indicated above, the process of the present inventhe 7 tion may beused for the ll-fi-oxygenation of a variety of concerned'with theintroduction of an hydroxyl group at The preparation of biologicallyactive steroid compounds, such as cortisone and compound F, isfraughtwith many great difficulties. One of the most difilcult problemsis the introduction of oxygen atoms at essential positions in thesteroid nucleus, particularly at the ll-position of this nucleus.Compound S is available by known synthetic routes from variou naturallyoccurring, relatively cheap, steroid starting materials, such as thevegetable-type steroid compounds. Compound F, on the other hand, isconsiderably more diflicult to obtain and is a very valuable compound,particularly useful in the treatment of rheumatoid arthritis and certainother conditions of the human body. Any process whereby compound S maybe converted to compound F in good yield and without undue expense is oftremendous value to the pharmaceutical industry and to the public ingeneral.

Methods have previously been reported for converting compound S tocompound F by means of organisms entirely different than those describedbelow for use in the present process. In U. S. Patent 2,658,023 there isdescribed the use of Curvularia organisms, and in U. S. Patent2,765,258, the use of Trichothecium. In U. S. Patent 2,602,769 there isdescribed the use of Cunninghamella blakesleeana (of the orderMucorales) and in a publication appearing in the Journal of the AmericanChemical Society (vol. 74, p. 2381 (1952)) a process utilizingStreptomyces fradiae is described. A great number of microorganisms hasbeen tested for their effective ness in converting compound S tocompound F. Most of these gave no indication of such llp-hydroxylation.

It has now been found that organisms of the genus Spondylocladium givestrikingly favorable results, allowing the preparation of such productsas compound F in yields of 10-20% or more. Furthermore, the reactionscan be conducted under such conditions that the products may be isolatedwith relative ease and in high purity.

It has been found that by contacting a steroid compound, in particularthose having a methylene group at the ll-position, with the oxygenatingactivity of the selected microorganisms, i. e. with the organisms themiselgles or with enzyme systems of the organisms, the sesteroid compoundswhich are unsubstituted in the ll-position of the nucleus. Various sidechains may be present at the 17-position of the nucleus and keto orhydroxyl or ester groups may be present. The steroid compounds used assubstrates for the reaction may also hear carbon to carbon double bondsat various points of the nucleus, such as the 3,4- or 5,6-p0sition. Itshould be realized that the yield of oxygenated product will vary tosome extent with the nature of the steroid compound used as startingmaterial, with the particular strain of organism used, and with theconditions employed for the reaction (i. e. temperature, time, pH,nutrient medium, time at which the compound is added to themicroorganism, etc.). Furthermore, a given oxygenating microorganism ofthe preferred species may show appreciable variation in its elfect uponvarious steroid compounds, that is, a good yield of the correspondingll-floxygenated derivative may be obtained with the use of a givenoxygenating strain and a specific steroid compound, whereas a secondsteroid compound used under otherwise exactly identical conditions maygive only a moderate or poor yield of the desired compound. Variousmethods may be used in the evaluation of the products produced by theseprocesses. For instance, if a steroid compound with a suitable sidechain is used, the proportion of the product produced may be evaluatedby determination of the eifect on adrenalectomized mice or upon theeosinophil count of experimental animals. Furthermore, the pure productsproduced by the hydroxylation reaction may be isolated as describedbelow.

The effectiveness of a chosen microorganism for the process of thisinvention may be determined by cultivating the organism in a suitablenutrient medium containing carbohydrates, salts, cources or organicnitrogen and so forth. The steroid compound as a solid or as solution ina suitable solvent, for example, acetone or ethanol, is added to thecultivated microorganism under sterile conditions and the mixture isagitated and aerated in order to bring about the growth of themicroorganism and oxygenation of the steroid substrate. The steroid maybe added when the medium is seeded under sterile conditions with aculture of the microorganism or after growth of the organism isestablished. In some cases it may be found advisable to add the steroidcompound after growth of the microorganism has been established in thenutrient medium under aerobic conditions. This is particularly true if,during the initial stages of growth of the microorganism, there is atendency to produce undesired by-products from the steroid substrate.The acetate or other ester of a steroid may be used in place of thealcohol itself, although this may sometimes lead to an appreciablylowered yield of hydroxylated product. Alternatively, enzymepreparations from the growth of a suitable oxygenating organism of thegenus may be used for conducting the process. A further, most usefulmethod is one in whichthe microorganism is grown on a suitable nutrientmedium under aerobic conditions in the absence of the steroid. Themycelial growth may then be filtered from the broth and may, if desired,be washed with distilled water. The mycelium is then suspended indistilled water containing the steroid substrate. Agitation of themixture and aeration is continued for a period of from about 12 to 48hours after which the products of the reaction are recovered. Thisprocess has the advantage of ease of recovery of the steroid compound,since the various nutrient materials originally used to obtain growth ofthe microorganism are now absent as well as the various materialsexcreted by the growing organisms during the initial period. With somestrains of the genus even better total yields of oxygenated products areobtained by this method than is the case when the steroid is added atthe beginning or at an intermediate period directed to the wholefermentation broth. Other methods familiar to enzyme chemists may beutilized for conducting the present oxygenating process. The proportionof prod ucts and the rate of oxygenation, as well as the nature of thelay-products formed, may vary depending on the use of the wholefermentation broth or of the isolated washed mycelium.

In general a concentration of not greater than one to two percent byweight of the total weight of substrate, for instance, the compoundS-type material, is used in conducting this process, although sometimesother concentrations may be found to be more favorably used. Since thesolubility of the starting material in water is quite limited, an excessof the material may be slowly converted to the oxygenated product.However, the state of subdivision of the steroid when added to theoxygcnating system, i. e. growing microorganism or enzyme system, doesnot seem to greatly affect the yield and nature of the products underotherwise identical conditions. If a water-miscible solvent solution ofthe steroid compound is added to the aqueous fermentation system, thesteroid is generally precipitated in finely divided form in the presenceof a large excess of water. This does not seemto appreciably improve therate of reaction as compared to the addition of dry, relatively largecrystals of the steroid.

After completion of the oxygenation process, the product may berecovered from the mixture by extraction with a suitablewater-immiscible solvent. Chlorinated lower hydrocarbons, kctones, andalcohols are useful. These include chloroform, methylene chloride,trichlorethane, ethylene dichloride, and so forth. The use of hotethylene dichloride, i. e. at from about 40 to about 80 C., isparticularly favored for the extraction of the steroid products. Theextract of product and unreacted starting material may be concentratedto a small volume or to dryness to obtain a solid product. Purificationof the product may be accomplished in several ways. Most useful is theseparation by means of chromatography of the product from the startingmaterial and from other products, such as more highly oxygenatedmaterials that may be formed during the reaction. Adsorbents such assilica gel or other suitable adsorbents are particularly useful for thispurpose. It has been found that a column prepared from a mixture ofsilica gel and a lower alcohol, especially ethanol, is particularlyuseful for the separation of the steroid starting materials. The steroidmixtures may be applied to columns of adsorbents such as silica gel inconcentrated chloroform or methylene chloride solution. The column maythen be washed with additional amounts of the solvent to remove suchimpurities as fats and pigments. The adsorbed mixture then is separatedby the gradual addition of a mixture of the solvent together with asmall percentage, for example l to 5%, of a lower alcohol (methanol,ethanol, etc.). The materials may be separated and the separatedcompounds gradually eluted from the column by utilization of a mixtureof solvents of gradually increasing polarity; for instance, a mixture ofmethylene chloride and a minor, gradually increasing amount of ethanolis very useful.

Fractions of the eluted material from chromatographic columns may bechecked for the nature of the product by subjecting small portions ofthe solutions to chromatography on paper. Methods which are particularlyuseful for conducting this type of separation and analysis are describedin detail in U. S. Patent 2,602,769, issued on July 8, 1952, to H. C.Murray et al., and in a publication by Shull et al. in the Archives ofBiochemistry, vol. 37, p. 186 (1952). This method is also very usefulfor evaluating new strains of microorganisms to determine theirusefulness in the process of this invention. The fermentation may beconducted on a small scale with the steroid as the substrate and thewhole extract of the fermentation mixture may be concentrated andsubjected to paper chromatography. By utilizing known samples of thesteroid, e. g. compound S, compound F and other related products, forcomparison, it is possible to determine whether the chosen microorganismis suitable for the present process.

Descending paper chromatograms utilizing paper treated with a 35%solution of propylene glycol in acetone or methanol and developed with amixture of 78 volumes of toluene and 22 volumes of dioxane may beutilized for the rapid evaluation of various strains of the preferredmicroorganisms. Such a separation can be completed in as little as threehours and the paper ehromatogram, after being dried, may be examinedunder ultraviolet light in a fluorescent scanner, such as that of Hainesand Drake (Federation Proceedings 1950. vol. 9, p. to determine theposition of the various materials by their fluorescence. The zones inwhich the various substances occur may be marked and cut from the sheetor strip of paper. The material may then be eluted with a solvent suchas ethanol and obtained as practically pure solid material byevaporation. A quantitative analysis of such a mixture may beaccomplished in this manner. The amount of the isolated products may bedetermined, for example, by measuring the ultraviolet absorption ofsolutions of the materials. Particularly useful is the absorptioncharacteristics of these compounds when dissolved in concentratedsulfuric acid.

After separation of the reaction products by column chromatography, thedesired fractions may be combined and concentrated to a small volume.The product may then be crystallized from a suitable solvent such asethyl acetate. One product prepared by application of the presentprocess has been compared with samples of authentic compound F and hasbeen found to be identical in all respects. Oorticosterone prepared bythe process of this invention has also been compared with a standardsample and shown to be identical with it.

The following examples are given by way of illustration and are notintended as a limitation of this invention. Indeed, as many apparentlywidely different embodiments of the present invention may be madewithout departing from the spirit and scope hereof, it is to beunderstood that the invention is only limited as defined in the appendedclaims.

Example I A culture of Spondylocladium auslrale was grown in flaskscontaining the following sterile medium:

Distilled water, adjusted to pH 7.0 with potassium hydroxide.

One hundred milliliters of this inoculum were added under sterileconditions to two liters of the following medium:

This mixture was adjusted to pH 7 with sulfuric acid and 0.25% ofcalcium carbonate was added before the mixture 'was sterilized. Theinoculated medium was aerated at the rate of about one-half to onevolume of air per volume of solution per minute at 27 to 28 C. for 24hours. During this time the mixture was stirred at the rate of about1700 revolutions per minute. One-half gram of compound S in the form ofthe alcohol was dissolved in 20 milliliters of 95% ethanol. The solutionwas added to the fermentation mixture under sterile conditions. Thereaction was then continued for a further 24 hours under exactly thesame conditions as described above.

The whole fermentation mixture was removed from the fermentation vessel.The mixture was extracted twice with an equal volume of ethylenedichloride at 70 C. The extracts were combined and evaporated todryness. The dry solids were dissolved in a small volume of methylenechloride and the solution was added to a column of silica gel. Thesilica gel column had been prepared previously by treating each gram ofsilica gel with one milliliter of 95% ethanol. This mixture wassuspended in methylene chloride and poured into a chromatographiccolumn. After the steroid mixture had been introduced into the column itwas washed with several portions of methylene chloride to remove fatsand pigments. The column was then developed by adding a mixture of 97volumes of methylene chloride and 3 volumes of ethanol. The eluate wasdivided into a series of small fractions. Portions of these wereanalyzed by means of the paper chromatographic system described aboveand those frac tions containing the same compound were combined. It wasfound that the first material leaving the column was unreacted compoundS. This material is recoverable and may be reused. The second materialleaving the column was an unidentified steroid. The third materialleaving the column was recovered and shown to be compound F. By removingthe solvent from the combined fractions con taining the compound F,there was obtained a yield of 12i0% of a dry product which may bereadily further puri- Example II A culture of Spondylocladium xylogenumwas cultivated on the same medium described in Example I under aerobicconditions. The mycelium from two liters of such a mixture obtainedafter 22 hours of growth was filtered, washed with a small volume ofdistilled water and then suspended in two liters of distilled water.One-half gram of compound S was added to the mixture. This preparationwas stirred and aerated at the rate of one-half volume of air per volumeof mixture per minute for 16 hours. The mixture was then extracted withone-half volume of chloroform three times. The combined chloroformextracts were concentrated to a small volume and the mixture of steroidswas purified by chromatography on silica gel. lure compound F wasobtained.

Example 1H Each of the or or the above examples was grown as describedin Evample I, except that in lieu of compound S the following steroidreactants containing 18 to 21 carbon atoms were employed:

Testosterone Progesterone o-dehydroprogesterone Androstenedionel7-hydroxyprogesterone Pregnenolone 3-keto-pregnane-20-ol3-keto-allopregnane-20-ol 1 A -pregnene-17a,21-diol-3,20-dione A-pregnadiene-17a,2l-diol-3,20-dione In each instance the correspondingllfl-hydroxylated product was obtained, as expected.

A strain of the organism S. australe has been deposited at the AmericanType Culture Collection under the number ATCC 12728. A strain of theorganism S. xylogenum has been deposited in the same collection underthe number ATCC 12727.

What is claimed is:

1. A process for the llfi-hydroxylation of a steroid rated from thebroth and the compound is contacted with an aqueous suspension of themycelium.

4. A process for the conversion of compound S to compound F, whichcomprises contacting compound S with the oxygenating activity of anorganism chosen from the genus Spondylocladium.

5. The process of claim 4 wherein the organism is Spondylocladiumaustrale.

6. The process of claim 4 wherein the organism is Spondylocladiumxylogenum.

7. A process for the preparation of compound F, which comprisescultivating an organismchosen from the genus Spondylocladium in anaqueous nutrient medium under aerobic conditions until substantialgrowth is obtained, then separating the mycelium from the broth, andtheneafter contacting compound S 'withan aqueous suspension of suchmycelium.

References Cited in the tile of this patent UNITED STATES PATENTS2,649,400 Murray et al Aug. 18, 1953 2,658,023 Shull et al. Nov. 3, 19532,765,258 Shull et al. Oct. 2, 1956 2,777,843 Chimerda et al Jan. 15,1957 OTHER REFERENCES Mycologia, Manuscript, any issue, Lancaster Press,

1. A PROCESS FOR THE 11B-HYDROXYLATION OF A STERIOD COMPOUND HAVING AMETHYLENE GROUP AT THE 11-POSITION AND CONTAINING 18 TO 21 CARBON ATOMS,WHICH COMPRISES CONTACTING SAID STEROID COMPOUND WITH THE OXYGENATINGACITIVITY OF AN ORGANISM CHOSEN FROM THE GENUS SPONDYLOCLADIUM.